Recombinant α-galactosidase a therapy for Fabry disease

ABSTRACT

Fabry disease results from an X-linked deficiency in the enzyme α-galactosidase A. The present invention is directed to recombinant human α-galactosidase A and provides baculovirus expression vectors and recombinant virus that provide stable expression of extracellular and intracellular levels of this enzyme in an insect cell culture. The recombinant-derived enzyme can be used in enzyme replacement therapy to treat Fabry patients. Composition useful in therapeutic administration of α-galactosidase A are also provided.

This application is a divisional of U.S. patent application Ser. No.08/155,734, filed Nov. 22, 1993, now U.S. Pat. No. 5,658,567, which, inturn, is a divisional of U.S. patent application Ser. No. 07/951,594,filed Sep. 28, 1992, abandoned, which in turn is a divisional of U.S.patent application Ser. No. 07/520,312 filed May 7, 1990, now U.S. Pat.No. 5,179,023 which is a Continuation-in-Part of U.S. patent applicationSer. No. 07/328,421 filed Mar. 24, 1989, abandoned.

FIELD OF THE INVENTION

Fabry disease results from an X-linked deficiency in the enzymeα-galactosidase A. The present invention is directed to recombinanthuman α-galactosidase A and provides baculovirus expression vectors andrecombinant virus that provide stable expression of extracellular andintracellular levels of this enzyme in an insect cell culture. Therecombinant-derived enzyme can be used in enzyme replacement therapy totreat Fabry patients. Compositions useful in therapeutic administrationof α-galactosidase A are also provided.

BACKGROUND OF THE INVENTION

Fabry disease is an X-linked inborn error of metabolism resulting from adeficiency of the lysosomal enzyme, α-galactosidase A. Deficiency ofα-galactosidase A results in the accumulation of its majorglycosphingolipid substrate, globotriaosylceramide and relatedglycolipids with terminal α-galactosidic linkages. Progressive substratedeposition, especially in the plasma and vascular endothelium, leads toischemia and infarction with early demise due to vascular disease of theheart and kidney.

Fabry diseases is one of approximately 30 lysosomal storage diseasesknown to affect humans. Each of these diseases result from an inheritedtrait which affects the levels of enzymes in the lyosome. Tay-Sach'sdisease and Gauucher disease are members of this group of diseases.Since specific pathways for the uptake of these other lysosomal enzymesalso exist, enzyme replacement therapy is expected to be effective inFabry disease and could logically be expected to be successful in theseother diseases as well. Although these diseases are individually rare,(e.g., several thousand patients with Fabry disease are known to occurworld wide, i.e., 1 to 40,000), as a group this class of diseasesaccounts for a significant fraction of all inherited diseases.

Several lines of evidence suggest that enzyme replacement therapy may bebeneficial for patients with Fabry disease. For example, it has beendemonstrated in cell cultures of fibroblasts obtained from patients withthis disease that enzyme present in the culture medium is specificallytransported to lysosomes. Clinical trials of enzyme replacement therapyhave been reported for patients with Fabry disease using infusions ofnormal plasma (Mapes et al., 1970, Science 169: 987-989);α-galactosidase A purified from placenta (Brady et al., 1973, New Eng.J. Med. 279: 1163); or α-galactosidase A purified from spleen or plasma(Desnick et al., 1979, Proc. Natl. Acad. Sci. USA 76: 5326-5330). In onestudy (Desnick et al.) intravenous injection of purified enzyme resultedin a transient reduction in the plasma levels of the substrate,globtriaosylceramide. However, due to the limited availability of thehuman enzyme obtained from human sources, insufficient quantities wereavailable for further study.

The α-galactosidase A enzyme is a lysosomal enzyme which hydrolyzesglobotriaosylceramide and related glycolipids which have terminalα-galactosidase linkages. It is a 45 kDa N-glycosylated protein encodedon the long arm of the X chromosome. The initial glycosylated forms(Mr=55,000 to 58,000) synthesized in human fibroblasts or Chang livercells are processed to a mature glycosylated form (Mr=50,000). Themature active enzyme as purified from human tissues and plasma is ahomodimer. (Bishop et al., 1986, Proc. Natl. Acad. Sci. USA 83:4859-4863).

A human liver cDNA for α-galactosidase A was identified in a gt11expression library (Calhoun et al., 1985, Proc. Natl. Acad. Sci. USA 82:7364-7368), and its sequence reported by Bishop et al. The original cDNAisolated by Calhoun et al. encoded the mature amino acid sequence ofα-galactosidase A but did not contain the complete signal peptidesequence of the precursor form.

The partial cDNA clone was used to construct an E. coli expressionvector by placing the α-galactosidase A coding sequence under control ofthe trp promoter (Hantzopoulos et al., 1987, Gene 57: 159-169). Thelevel of expression of active enzyme was sufficient to support E. coli.growth using an α-galactoside substrate as the sole carbon source;however, increased levels of the 45 kDa protein were not detected inCoomassie blue-stained gels upon trp promoter induction nor wasbiological activity detected in vitro.

A genomic clone was later isolated which carried the promoter and firstexon of the protein including the full signal peptide (Quinn et al.,1987, Gene 58: 177-188). Clone of full length cDNAs of a precursorα-galactosidase A from human fibroblasts were reported (Tsuji et al.,1987, Eur. J. Biochem. 165: 275-280) and used to obtain transientexpression of the enzyme in monkey COS cells. The level of enzymeactivity reported was only about 40% above background.

Hence one long standing need of the prior art is to provide largequantities of active human α-galactosidase A, especially for use inenzyme replacement therapy. To achieve this goal, a full length cDNA ofhuman α-galactosidase A is needed which can be incorporated into anexpression vector under control of a strong promoter. Furthermore, thisvector should provide stable expression of the cDNA and use a hostsystem in which the processing and glycosylation may occur. Finally,biologically active enzyme must be produced. One such expression vectoris provided by the baculovirus expression system of the presentinvention.

Baculoviruses infect Lepidopterm insects and have proven useful asrecombinant expression vectors (Smith et al., 1983, Mol. Cell. Biol. 3:2156-2165; Luckow et al., 1988, Bio./Technology. 6 :47-55). The latterof these two references provides a detailed description of the availablebaculovirus vectors, methodology for their use and a list of proteinswhich have been expressed in this system. The particular advantage ofbaculovirus expression systems are very high levels of production (1 mgto 500 mg of protein per liter of culture have been reported),glycosylation and processing of the so-produced proteins. The earlybaculovirus expression vectors employ a strong promoter for anonessential gene, the polyhedrin gene. To facilitate cloning, a DNAsequence encoding several restriction endonuclease sites had beeninserted into the polyhedrin promoter (Luckow, et al., 1988,Bio/Technology 6 :47-55). Subsequently, it was discovered that thisgenetically engineered promoter was less effective (2-1000 fold) thanthe wild type polyhedrin promoter (Page, 1987, Nuc. Acids Res. 17 454;Ooi et al., 1989, J. Molec. Biol. 210 721-736). The present inventionthus employs both the genetically altered polyhedrin promoter and thewild type polyhedrin promoter to generate expression systems providinglarge amounts of active α-galactosidase A.

SUMMARY OF THE INVENTION

The present invention is directed to replicable expression vectors thatexpress the human α-galactosidase A enzyme. These vectors arebaculovirus derivatives which have been genetically engineered tocontain a full length cDNA or recombinant DNA encoding the precursorform of α-galactosidase A. The cDNA, for example, is operably linked tothe baculovirus polyhedrin promoter so that the promoter directsexpression of biologically active, human α-galactosidase A under theappropriate conditions. This promoter can be either a geneticallyengineered polyhedrin promoter with an insertion of DNA encoding severalrestriction endonuclease sites or wild type polyhedrin promoter.Transformant microorganisms or cell cultures carrying these vectors arealso provided by the present invention.

Another aspect of the present invention provides recombinantbaculoviruses having a gene sequence for the precursor form of a humanα-galactosidase A enzyme and which express high levels of said enzyme.

A further aspect of this invention provides homogenous recombinant humanα-galactosidase A and antibodies directed against this enzyme ordirected against any active fragment or derivative of the enzyme.

A still further aspect of the present invention provides pharmaceuticalcompositions comprising an effective amount of recombinant humanα-galactosidase A, or an active derivative thereof, and apharmaceutically acceptable carrier. These compositions are useful intreating diseases associated with a deficiency of this enzyme,especially Fabry disease.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 depicts the human α-galactosidase cDNA features encoded onM13.L21.1 (top), the restriction enzyme recognition sites in thepolylinker region of pSPR1 (middle) and a partial restriction map andsome of the features of pAc373, a baculovirus transfer vector (bottom).

FIG. 2 depicts the differences in DNA sequence between transfer vectorspCC4 (top) and pCC14 (bottom). Elimination of 93 bp from the promotersequences of pCC4 (underlined) and replacement with 7 bp (underlined) inpCC14 increases α-galactosidase A protein expression at least 2-10 fold.The ATG start codon (box) is indicated in both sequences.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to replicable expression vectors whichare used to express human α-galactosidase A. These vectors arerecombinant baculoviruses having an α-galactosidase A cDNA under controlof the Autographa californica nuclear polyhedrosis virus (AcMNPV)polyhedrin promoter or any other strong baculovirus promoter capable ofexpressing foreign genes. An insect cell culture, for example Spodopterafrugiperda line Sf9, infected with this recombinant virus expressesα-galactosidase A both intracellularly and extracellularly at unexpectedlevels.

Baculovirus expression vectors and their use are reviewed in Luckow etal. The baculoviruses have double-stranded circular DNA genomesapproximately 130 kb in length. These viruses have a wide host rangeinfecting about 30 species of Lepidopterm insects. In particular, AcMNPVinfection of Spodoptera frugiperda cell cultures produce very highlevels of a protein known as polyhedrin which may account for 50% of thecellular protein. Polyhedrin is a nonessential viral gene which can bereplaced by a foreign gene in order to express that gene under controlof the strong polyhedrin gene promoter. The recombinant viruses are thenused to infect a S. frugiperda cell culture to produce the desiredprotein. A particular advantage of this system is its similarity tohigher eukaryotes with regard to protein modification, processing andtransport. Thus, recombinant-derived eukaryotic proteins will beprocessed and glycosylated in a manner important for biologicalactivity.

To produce a recombinant baculovirus which expresses a foreign gene,that gene is inserted into a transfer vector downstream of thepolyhedrin promoter. The transfer vector is a plasmid which has beengenetically engineered to contain DNA flanking the polyhedrin gene aswell as convenient restriction enzyme recognition sites adjacent to thepolyhedrin promoter or the polyhedrin gene initiation codon.Alternatively, the engineered polyhedrin promoter can be mainpulated,e.g. its sequences changed to the original wild type or otherexpression-effecting sequence, to further increase expression levels.The transfer vector and viral DNA are used to co-transfect insect cellcultures. Homologous recombination will occur between the transfervector and viral DNA during transfection and the native polyhedrin geneis replaced by the engineered polyhedrin promoter and foreign gene onthe transfer vector. The viral progeny are then screened for occlusionnegative (Occ⁻) virus, the phenotypic characteristic for loss of thepolyhedrin gene. Occlusions are particles that appear in the cytoplasmof an infected cell and consist of virus particles embedded in acrystalline lattice of the polyhedrin protein.

The recombinant baculovirus expression vectors of the present inventionare made by inserting a cDNA or a recombinant DNA for the precursor formof human α-galactosidase A downstream of the polyhedrin promoter in anbaculovirus transfer vector, for example the AcMNPV transfer vectorpAc373. The precursor form consists of the signal peptide and the matureα-galactosidase A. This transfer vector and many other suitable vectorsare described in Luckow et al. In pAc373 there is a unique BamHI sitebetween the polyhedrin promotes and coding region into which a DNAfragment containing the α-galactosidase A gene may be cloned.

A DNA fragment encoding the full α-galactosidase A coding sequenceincluding the complete signal peptide is found on the EcoRI fragmentfrom M13.L21.1. Construction of this M13 derivative is described inHantzopoulos (1987, Ph.D. Thesis). To put BamHI ends on this EcoRIfragment, the fragment is cloned into the polylinker of pSPR1 (Krystalet al. (1986) Proc. Natl. Acad. Sci. USA 83: 2709-2713). Because thepSPR1 polylinker is composed of two smaller polylinkers cloned head tohead at a unique EcoRI site, it provides a convenient way to introduce avariety of restriction enzyme sites on the ends of any EcoRI fragment.The BamHI fragment of the pSPR1 derivative containing theα-galactosidase A gene is then cloned into the unique BamHI site inpAc373. The resulting transfer vector, designated pCC4, contains thecomplete α-galactosidase A cDNA in the proper orientation relative tothe polyhedrin promoter.

To increase expression of α-galactosidase A the genetically engineeredpolyhedrin promoter in pCC4 was replaced by the wild type polyhedrinpromoter (Ooi, et al., 1989, J. Molec. Biol. 210: 721-736). pCC4 DNA wascut with EcoRV and BssHII restriction endonucleases and the 213 bpEcoRV/BssHII fragment was discarded. This deletes 178 bp upstream and 35bp downstream of the α-galactosidase A ATG start codon of pCC4 (See FIG.2). To reconstruct the wild type polyhedrin promoter, and to replace theportion of the coding region lost by this deletion, 4 overlappingoligonucleotides were synthesized, annealed together and ligated intothe EcoRV/BssHII cut pCC4 DNA. This new α-galactosidase A transfervector is called pCC14. The pCC14 DNA sequences which replace thedeleted DNA sequences in pCC4 are shown in FIG. 2 (differences in DNAsequence are underlined). These manipulations do not alter the ATG startcodon or the coding region. The only alteration is in the length ansequence of DNA lying between the transcription start signals and theATG start codon (7 bp in pCC14 replace 93 bp in pCC4). The DNA sequenceof the pCC14 promoter was confirmed by DNA sequencing. Proteinexpression from pCC14 can increase up to 1000-fold for α-galactosidaseA, relative to expression from pCC4.

To prepare a recombinant baculovirus derivative carrying the humanα-galactosidase A gene as a cDNA, a transfer vector carrying that DNA,for example pCC4 or pCC14, and AcMNPV DNA, are used to cotransfect aninsect cell culture line, preferably S. frugiperda cell line Sf9. Thetransfer vector and viral DNA undergo recombination between thehomologous baculovirus sequences which flank the α-galactosidase A geneand thereby produce the desired recombinant baculovirus. To identifythis recombinant baculovirus, the viral progeny from the transfectionare plated on Sf9 cells and the plaques are screened to find Occ⁻viruses. Methods for distinguishing Occ⁻ plaques from Occ⁺ plaques aredescribed in Summers et al. (1987) A Manual of Methods for BaculovirusVectors and Insect Cell. Culture Procedure, Texas AgriculturalExperiment Station, Bulletin No. 1555. In addition, this publicationprovides protocols for all of the above described manipulations andaccordingly is incorporated herein by reference.

The human α-galactosidase A cDNA encoding the precursor form of thispolypeptide has the following nucleotide sequence and amino acidsequence:

ATBCABCTBAFBRACC M  D  L  R  K  CABAACTACATCTBEBCTBCBCBCTTBCBCTTCGCTTCCTBGCCCTCBTTTCCTBBBACATCCCTBBBBCTABABCA−1P  E  L  H  L  B  C  A  L  A  L  R  F  L  A  L  V  S  W  D  I  P  B  A  R  A −1CTG CAC AAT CCA TTC CCA AGG ACG CCT ACC ATG GGC TGG CTG CAC TGG GAG CGC TTC ATG TCC AAC CTT GAC TCC CAG GAA GAG CCA CAT90Leu Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp Glu Arg Phe Met Cys Asn Leu Asp Cys Gln Glu Glu Pro Asp30                                                                                                Ser         ArgTCC TGC ATC AGT GAG AAG CTC TTC ATG GAG ATG GCA GAG CTC ATG GTC TCA GAA CCC TCC AAG CAT CCA CCT TAT GAG TAC CTC TGC ATT180Ser Cys Ile Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met Val Ser Glu Gly Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile60                 SerGAT GAC TGT TGG ATG CCT CCC CAA AGA GAT TCA GAA CCC AGA CTT CAG CCA GAC CCT CAG CGC TTT CCT CAT GGG ATT CGC CAG CTA GCT270Asp Asp Cys Trp Met Aln Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln Ala Asp Pro Gln Arg Phe Pro His Gly Ile Arg Gln Leu Ala90AAT TAT CTT CAC AGC AAA GGA CTG AAG CTA GGG ATT TAT GCA GAT GTT GGA AAT AAA ACC TCC GCA GGC TTC CCT GGG AGT TTT GGA TAC360Asn Tyr Val His Ser Lys Glu Leu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala Gly Phe Pro Gly Ser Phe Gly Tyr120TAC GAC ATT GAT GCC CAG ACC TTT GCT GAC TGG GGA GTA CAT CTG CTA AAA TTT GAT GGT TGT TAC TCT GAC AGT TTG GAA AAT TTG GCA450Tyr Asp Ile Asp Ala Gln Thr Phe Ala Asp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp Ser Leu Glu Asn Leu Ala150CAT GGT TAT AAG CAC ATG TCC TTG GCC CTG AAT AGG ACT GGC AGA ACC ATT GTG TAC TCC TGT GAG TGG CCT CTT TAT ATG TGG CCC TTT540Asp Gly Tyr Lys His Met Ser Leu Ala Leu Asn Arg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp Pro Leu Tyr Met Trp Pro Phe180CAA AAG CCC AAT TAT ACA GAA ATC CGA CAG TAC TCC AAT CAC TGG CGA AAT TTT GCT GAC ATT GAT GAT TCC TGG AAA AGT ATA AAG ACT630Gln Lys Pro Asn Tyr Thr Glu Ile Arg Gln Tyr Cys Asn Met Trp Arg Asn Phe Ala Asp Ile Asp Asp Ser Trp Lys Ser Ile Lys Ser210                                                                                    AsnATC TTG GAC TGG ACA TCT TTT AAC CAG GAG AGA ATT GTT CAT GTT GCT GGA CCA GGG GGT TGG AAT GAC CCA GAT ATG TTA GTG ATT GGC720Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu Arg Ile Val Asp Val Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val Ile Gly240AAC TTT GGC CTC ACC TGG AAT CAG CAA GTA ACT CAG ATG GCC CTC TGG CCT ATC ATG GCT GCT CCT TTA TTC ATG TCT AAT GAC CTC CGA810Asn Phe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln Met Ala Leu Trp Ala Ile Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg270                                                                                                    AlaCAC ATC AGC CCT CAA GCC AAA GCT CTC CTT CAG GAT AAG GAC GTA ATT GCC ATC AAT CAG GAC CCC TTG GGC AAG CAA GGG TAC CAG CTT900His Ile Ser Pro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp Val Ile Ala Ile Asn Gln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu300                                                        Arg                 GluAGA CAG GGA GAC AAC TTT GAA CTG TGG CAA CGA CCT CTC TCA GGC TTA CCC TGG GCT GTA CCT ATG ATA AAC AGG CAG CAC ATT GGT CCA990Arg Gln Gly Asp Asn Phe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val Ala Met Ile Asn Arg Gln Glu Ile Gly Gly330     Leu                         Gly Ser LysCCT CGC TCT TAT ACC ATC GCA GTT GCT TCC CTG GGT AAA GGA GTG GCC TGT AAT CCT GCC TGC TTC ATC ACA CAG CTC CTC CCT GTG AAA1080Pro Arg Ser Tyr Thr Ile Ala Val Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro Ala Cys Phe Ile Thr Gln Leu Leu Pro Val Lys360AGG AAG CTA GGG TTC TAT GAA TGG ACT TCA AGG TTA AGA AGT CAC ATA AAT CCC ACA GGC ACT GTT TTG CTT CAG CTA GAA AAT ACA ATG1170Arg Lys Leu Gly Phe Tyr Glu Trp Thr Ser Arg Leu Arg Ser His Ile Asn Pro Thr Gly Thr Val Leu Leu Gln Leu Glu Asn Thr Met390 CAG ATG TCA TTA AAA GAC TAA CTT TAAAAAAAAAAAAAA 1209Gln Met Ser Leu Lys Asp Leu Leu Ter 398

Hence, the recombinant baculoviruses of the present invention providehigh level, stable expression of biologically active humanα-galactosidase A. The stable expression of high levels of biologicallyactive human α-galactosidase A is unique to the present invention. Suchexpression and activity is particularly high in insect cell culturesinfected with these recombinant baculoviruses in this preferredexpression system, especially Sf9 cells. Recombinant α-galactosidase Aactivity is present in the culture medium as well as the cells, with themajority of the activity found in the culture medium. Extracellular,recombinant enzyme activity is thus achieved by the present invention.Furthermore, the expression system of the present invention providesglycosylated α-galactosidase A which is biologically active. Theα-galactosidase A activity can be measured using a fluorescentsubstrate, 4-methylumbelliferyl-α-D-galactopyranoside, for example.

Another aspect of the present invention provides homogeneous recombinanthuman α-galactosidase A. Homogeneous preparations are particularlyuseful for enzyme replacement therapy of Fabry disease and any otherdisease resulting from a deficiency of α-galactosidase A. Recombinantproduction of α-galactosidase A provides a plentiful source of activeenzyme which was heretofore unavailable from natural sources or otherrecombinant sources. Apparently, the increased activity relative to theE. coli expression system already described is due to glycosylation andprocessing of the enzyme neither of which occurred when theα-galactosidase was expressed in E. coli.

Purification of recombinant α-galactosidase A from the culture medium orthe intact cells, if desired, is achieved by conventional purificationmeans such as ammonium sulfate precipitation, column chromatography andthe like by following the enzymatic activity of the recombinantα-galactosidase A by the assay described herein.

One preferred scheme to purify recombinant α-galactosidase A produced bya baculovirus expression vector of the present invention involvesharvesting the culture supernatent when the α-galactosidase A activityis at a peak, typically about 48 to 72 hours after viral infection ofSf9 cells. The proteins in the supernatent are precipitated by ammoniumsulfate, dialyzed into an appropriate buffer and applied to aConcanavalin A-Sepharose (Pharmacia trademark) chromatography resin. Theresin is eluted with 0.1 M α-methylglucoside to remove contaminants andthen with 1 M α-methylglucoside to release the bound α-galactosidase Aactivity. After concentrating and dialyzing the eluate which containsthe activity, it is applied to Sephadex G-200 or a DEAE-Sephadex A-50(Pharmacia trademark) column. When applied to a DEAE column the activefractions are pooled and can be further purified on a hydroxylapatitecolumn to an estimated 95% homogeneity.

The recombinant α-galactosidase A produced by the baculovirus expressionsystem, e.g. in Sf9 cells, is also characterized by glycosylation. Inparticualr, the recombinant enzyme present in culture supernatants bindsto Con-A during the purification process, which indicates the presenceof α-D-glucosyl or α-D-mannosyl residues on the enzyme. Moreover,treatment of a hydroxylapatite pool obtained during purification, withN-glycanase produces a marked decrease in the apparent molecular weightof the enzyme, and converts a rather diffuse band to a faster migratingand more compact band. Treatment with endoglycosidase H results inslight but reproducible changes in the migration of the enzyme in SDSgels, indicating the presence of high mannose oligosaccharide. Theseresults are consistent with the presence of several glycoforms of therecombinant α-galactosidase A produced by insect cells, including bothhigh mannose and complex type carbohydrate side chains.

The present invention also provides polyclonal and monoclonal antibodiesto recombinant α-galactosidase A, especially from a human source. Theseantibodies are prepared by methods well known in the art. Extensiveprotocols for preparing, purifying, identifying, and use of monoclonalantibodies are found in Harlowe et al. (1988) Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 726p.,which is incorporated herein by reference.

Monoclonal antibodies are conveniently prepared by immunizing mice withhomogeneous or partially purified recombinant α-galactosidase A.Alternatively, fragments or active derivatives of α-galactosidase A maybe used for immunization. These fragments may be made by proteolyticdigestion and purified by conventional means. The enzyme derivatives maybe made by chemical modification or site-directed mutagenesis of thecloned α-galactosidase A gene or cDNA. Likewise, any of theseα-galactosidase A preparation are used to prepare polyclonal antibodiesin rabbits or other animals such as goat, sheep, rats or the like.Methods of identifying the desired antibody include ELISA assay usingany purified α-galactosidase A as the test material, Western orimmunoblotting against pure or partially pure α-galactosidase A, andother methods as described in Harlowe et al. The antibodies are usefulto affinity purify large quantities of α-galactosidase A, including therecombinant enzyme.

In a related embodiment, the present invention also contemplates thegene fusion of α-galactosidase A to homologous human proteins including,but not limited to, albumin, insulin and apoprotein B. Fusions may beconstructed to specific fragments of these genes that stabilize theα-galactosidase A and retain specific receptors for endocytosis. Thistechnique also contemplates the genetically altered versions of theproteins employed where, for example, it may be advantageous toeliminate the biological activity of the protein, e.g., insulin, whilemaintaining its intended efficacy in this context.

Another aspect of the present invention provides recombinant humanα-galactosidase A as a valuable therapeutic agent for treating diseasesresulting from deficiencies of this enzyme, especially Fabry disease, ina mammal by administering to said mammal an effective amount ofrecombinant α-galactosidase A or an active derivative or fragmentthereof for a time and under conditions sufficient to treat thedeficiency by increasing enzyme level.

A therapeutically effective amount of recombinant α-galactosidase A mayrange from about 50 to about 10,000 units enzyme activity per kg bodyweight per day. A unit of α-galactosidase A activity is as defined inCalhoun et al., with one unit corresponding to one nanomole of4-methylumbelliferyl-α-D-galactopyranoside hydrolyzed per hour at 37° C.

For example, the uptake of recombinant α-galactosidase A in normal andFabry fibroblasts can be examined to show that the recombinantlyproduced enzyme restores α-galactosidase A activity in cells lacking theenzyme. Restoration of function can be tested by addition of thepurified enzyme to the culture medium and the results (described indetail in Example 5) indicate that recombinant α-galactosidase A istaken up in a dose dependent fashion that produces an approximately 59%increase in enzyme levels in normal fibroblasts, and a completerestoration of enzyme levels in Fabry fibroblasts. These results arequite similar to those reported for the uptake of α-galactosidase Apurified from human sources (Mayes et al., 1982, Am. J. Hum. Genet. 34:602-610) and indicate that the recombinant α-galactosidase A produced inthis expression system is a valuable therapeutic agent for treatment ofFabry disease.

Accordingly, the subject invention contemplates treating Fabry diseaseor other diseases resulting from a deficiency of α-galactosidase A inmammals by administering a pharmaceutical composition containing apharmaceutically effective amount of recombinant α-galactosidase A or anactive fragment or derivative thereof. Additionally, a method fortreating Fabry disease (or other diseases characterized by this enzymedeficiency) in a mammal is contemplated in which a nucleic acid moleculeencoding α-galactosidase A is introduced into a cell in such a mannerthat said nucleic acid molecule is expressed intracellularly. By activefragments is meant any part of the enzyme which is derived from theintact whole enzyme and still retains biological activity. Likewise,derivatives of α-galactosidase mean enzymes which have been chemicallymodified or genetically engineered to effect minor changes, for exampleamino acid substitutions, which maintain biological activity. Suchexpression may be extrachromosomal in said cell or occur followingintegration into the genome of said cell. In this case where the enzymeis introduced into the cell via a nucleic acid, the nucleic acidmolecule may be carried to the cell and transferred into said cell by asecond nucleic acid molecule (e.g., various viruses). The first nucleicacid molecule is manipulated such that it contains the appropriatesignals for expression in the target cell. That is, in accordance withthe present invention, a method of treating Fabry disease in a mammal iscontemplated by administering a first nucleic acid molecule encodingα-galactosidase A. This nucleic acid is contained in a pharmacologicallyacceptable second nucleic acid carrier such that said first nucleic acidenters a target cell and is either maintained extrachromosomally orintegrated into the genome of said target. Thus, expression of the firstnucleic acid produces an effective amount of human α-galactosidase A.

The active ingredients of the pharmaceutical compositions comprisingrecombinant α-galactosidase A are contemplated to exhibit excellent andeffective therapeutic activity in replacing the enzymatic deficiencyfound in Fabry disease or other conditions resulting from thisdeficiency. Thus, the active ingredients of the therapeutic compositionsincluding recombinant α-galactosidase A exhibit enzymatic activity whenadministered in therapeutic amounts from about 0.1 ug to about 2000 ugper kg of body weight per day. The dosage regimen may be adjusted toprovide the optimum therapeutic response. For example, several divideddoses may be administered daily or the dose may be proportionallyreduced as indicated by the exigencies of the therapeutic situation. Adecided practical advantage is that the active compound may beadministered in a convenient manner such as by the oral, intravenous(where water soluble), intramuscular, intravenous, intranasal,intradermal, subcutaneous, or suppository routes. Depending on the routeof administration, the active ingredients of a recombinantα-galactosidase A-containing pharmaceutical composition may be requiredto be coated in a material to protect said ingredients from the actionof enzymes, acids or other natural conditions.

The active compounds may also be administered parenterally orintraperitoneally. Dispersions can also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases the form must be sterile and mustbe fluid to the extent that easy syringability exists. It must be stableunder the conditions of manufacture and storage and must be preservedagainst the contaminating action of microorganisms such as bacteria andfungi. The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. The preventions of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Coatings for α-galactosidase A preparations are useful to reducedegradation of the enzyme when administered as a therapeutic agent.Coatings also reduce the immunogenicity of the enzyme to help preventundesirable side effects of administering such a therapeutic agent. Aparticularly useful coating to provide these characteristics ispolyethylene glycol.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfilter sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and the freeze-dryingtechnique which yield a powder of the active ingredient plus anyadditional desired ingredient from previously sterile-filtered solutionthereof.

When recombinant α-galactosidase A is suitably protected as describedabove, the active compound may be orally administered, for example, withan inert diluent or with an assimilable edible carrier, or it may beenclosed in hard or soft shell gelatin capsule, or it may be compressedinto tablets, or it may be incorporated directly with the food of thediet. For oral therapeutic administration, the active compound may beincorporated with excipients and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. Such compositions and preparation should contain at least1% of active compound. The percentage of the compositions andpreparations may, of course, be varied and may conveniently be betweenabout 5 to about 80% of the weight of the unit. The amount of activecompound in such therapeutically useful compositions is such that asuitable dosage is obtained. Preferred compositions or preparationsaccording to the present invention are prepared so that an oral unitdosage form contains between about 10 ug and 1000 ug of active compound.

The tablets, troches, pills, capsules and the like may also contain thefollowing: A binder such as gum agragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; a sweetening agent such assucrose, lactose or saccharin or a flavoring agent such as peppermint,oil of wintergreen, or cherry flavoring. When the dosage form is acapsule, it may contain, in addition to materials of the above type, aliquid carrier. Various other materials may be present as coatings or tootherwise modify the physical form of the unit dosage. For instance,tablets, pills, or capsules may be coated with shellac, sugar or both. Asyrup or elixir may contain the active compound, sucrose as a sweeteningagent, methyl and propylparabens as preservatives, a dye and flavoringsuch as cherry or orange flavor. Of course, any material used inpreparing any dosage unit form should be pharmaceutically pure andsubstantially non-toxic in the amounts employed. In addition, the activecompound may be incorporated into sustained-release preparations andformulations.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Unit dosage form as used herein refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the novel dosageunit forms of the invention are dictated by and directly dependent on(a) the unique characteristics of the active material and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active material for the treatment ofdisease in living subjects having a diseased condition in which bodilyhealth impaired as herein disclosed in detail.

The principal active ingredient, especially, recombinant α-galactosidaseA, is compounded for convenient and effective administration inpharmaceutically effective amounts with a suitable pharmaceuticallyacceptable carrier in dosage unit form as hereinbefore disclosed. A unitdosage form can, for example, contain the principal active compound inamounts ranging from 10 ug to about 1000 ug. Expressed in proportions,the active compound is generally present in from about 10 ug to about1000 ug/ml of carrier. In the case of compositions containingsupplementary active ingredients, the dosages are determined byreference to the usual dose and manner of administration of the saidingredients.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like. The useof such media and agents for pharmaceutical active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

The following examples further illustrate the invention.

EXAMPLE 1 Construction of a Baculovirus Derivative to Express Humanα-galactosidase A

The clone that contains the complete coding region for the precursorform of human α-galactosidase A, M13.L21.1 (Hanztopoulos) was digestedwith EcoRI. All restriction enzyme digestions were performed accordingto manufacturers instructions, New England Biolabs. This EcoRI digestionwas ethanol precipitated arid mixed with EcoRI-digested pSPR1 inligation buffer. After ligation with T4 DNA ligase as recommended by themanufacturer (New England Biolab), recombinants were selected forampicillin resistance and restriction mapped to identify a pSPR1recombinant with the EcoRI fragment having the human α-galactosidase Acoding sequence. The pSPR1 derivative was then digested with BamHI andmixed with BamHI-digested pAc373, the baculovirus transfer vector whichhas a unique Bam III site 8 bp upstream from the ATG codon of thepolyhedrin gene coding region. After ligation, recombinants wereselected for ampicillin resistance, and restriction mapped to identifytwo clones having the pSPR1-derived BamHI fragment inserted into theBamHI site of pAc373 in opposite orientations. These clones are thetransfer vectors pCC4 which has the α-galactosidase A cDNA in theorientation properly aligned the polyhedrin promoter and pCC5 which hasthe cDNA in the opposite orientation. The vector pCC5 is a controlplasmid.

Transfer vector pCC4 or pCC5 were co-transfected with AcMNPV DNA into S.frugiperda Sf9 cells by the methods described in Summers et al. About 3%of the plaques were Occ⁻. Several recombinant viral clones were plaquepurified three times from the Occ⁻ clones. The resulting recombinantbaculoviruses from transfer vector pCC4 are designated AcCC1 and AcCC2.The control recombinant virus from transfer vector pCC5 is designatedAcCC3.

Restriction maps of M13.L21.1 and pAc373 are shown in FIG. 1 (top andbottom). The center portion of FIG. 1 depicts the polylinker region ofpSRP1.

EXAMPLE 2 Expression of Human α-Galactosidase A by RecombinantBaculovirus Vectors

To test for α-galactosidase A production, the recombinant viruses wereused to infect Sf9 cells. Cells were cultured and infections carried outas described in Summers et al. Exponential phase infected cells wereharvested, separated from the culture medium, and both the cells and theculture medium tested for α-galactosidase A activity. The wild typevirus, AcMNPV and mock infected cells were included as controls. Theactivity of α-galactosidase A was measured using the fluorescentsubstrate, 4-methylumbelliferyl-α-D-galactopyrassoside and is expressedas units (nanomoles per hour at 37° C.) present in the cells or culturemedium in a 25 cm² flask containing 5 ml of medium and approximately3×10⁶ cells.

Table 1 indicates that recombinant viruses AcCC1 and AcCC2 express highlevels of active recombinant human α-galactosidase A in the culturemedium and significant levels in the cells. In contrast, the controlviruses AcCC3, and AcMNPV as well as mock infections have relativelylittle enzyme activity. The recombinant viruses AcCC1 and AcCC2 expressabout 1000-fold higher levels of enzyme activity than the recombinantcontrol virus, AcCC3.

Further, since α-galactosidase A activity is found in the culturemedium, it suggests that the precursor form is properly processed to themature form.

EXAMPLE 3 Construction and Expression of Human α-Galactosidase A in aModified Baculovirus Vector

Transfer vector pCC4, containing the human α-galactosidase A cDNA clonedin proper orientation to the modified polyhedrin promoter derived frompAc373, was digested with EcoRV and BssHII (enzymes and used asrecommended by the manufacturer, New England Biolabs). The 213 bpEcoRV/BssHII fragment was discarded. To construct the wild typepolyhedrin promoter, 4 overlapping oligonucleotides were synthesized,annealed together and mixed with the large fragment of EcoRV/BssHII-cutpCC4 DNA in ligation buffer. After ligation with T4 DNA ligaserecombinants were transformed into E. coli and selected on ampicillinplates. Recombinants with the wild type polyhedrin promoter wereidentified by their inability to hybridize (Sambrook et al., 1989,Molecular Cloning, a Laboratory Manual 2 pp. 8.1-15.173) to a ³²Plabeled oligonucliotide probe derived from the 93 bp region of DNAdeleted from pCC4 (FIG. 2, top). To confirm that the selected clone hadthe proper DNA sequence of the wild type promoter, double strandedrecombinant DNA was sequenced using unique oligonucliotide primershomologous to sequences lying outside the altered region. Twos cloneswere identified with the correct sequence (FIG. 2 bottom). The modifiedpCC4 transfer vector was named pCC14.

After co-transfection of pCC14 and viral DNA into SF9 cells as describedin Example 2, virally infected cloned cells were tested forα-galactosidase A activity. The recombinant virus which has incorporatedpCC14 sequences is called AcCC4 and produce at least 2-10 fold moreα-galactosidase A activity than does the pCC4 containing virus AcCC1after on round of plaque purification. After three rounds of plaquepurification to yield a homogeneous preparation of AcCC4, theα-galactosidase A levels can increase up to about 1000 fold relative toAcCC1-derived enzyme levels.

EXAMPLE 4 Purification of Recombinant α-Galactosidase A and CarbohydrateCharacterization

To purify the recombinant human α-galactosidase A a modification of theprocedure of Calhoun, et al. (1985, Proc. Natl. Acad. Sci. USA 82,7364-7368) was used. The culture medium from SF9 cells infected withrecombinant virus was collected and the enzyme precipitated withammonium sulfate. Precipitated crude α-galactosidase A was dialyzed andapplied to a Concanavalin A-Sepharose (Pharmacia) column. The column waswashed with 0.1 M α-methylglucoside to remove contaminants andα-galactosidase A was eluted with 1 M α-methylglucoside. Afterconcentration using an Amicon (Danvers, Mass.) concentrator with a PM-10membrane and dialysis, the pool of active enzyme was applied to aDEAE-Sephadex A-50 (Pharmacia) column. The column was eluted with agradient of 10 to 200 mM NaCl and the active fractions were pooled,concentrated and dialyzed. These pooled fractions were applied to ahydroxylapatite column and α-galactosidase A was eluted with a gradientof 20 to 200 mM sodium phosphate, pH 6.5. The concentrated, dialyzedα-galactosidase A from this purification scheme was judged to be 95%homogeneous by SDS gel electrophoresis.

All of the recombinant enzyme bound to the Concanavalin A column,indicating that carbohydrate residues such as α-D-glucosyl orα-D-mannosyl were present on the enzyme. To further investigate theglycosylation of α-galactosidase A, aliquots of the purified enzyme wereseparately treated with two enzymes that remove selective carbohydratemoieties from the protein.

Recombinant endoglycosidase H (ICN Biomedicals, Lisle, Ill.; code number39-131) was dissolved in 27 mM sodium phosphate buffer, pH 6.5,containing 50% glycerol at a final concentration of 1.25 units/ml. Oneunit of enzyme hydrolyzes one mole of [³H]-dansyl-Asn(G1cNAC)₂ per minat 37° C. N-Glycanase (Genzyme, Boston, Mass.) was supplied as a 50%glycerol solution containing 250 units/ml. One unit of enzyme hydrolyzesone nanomole of [³H]-dansyl-fetuin glycopeptide per min at 37° C.Aliquots of the enzyme obtained from the hydroxylapatite pool containing14 μg of protein were adjusted to 0.5% NaDodSO₄, 0.1 M β-mercaptoethanoland boiled for 2 min. The samples were then diluted to either 250 mMsodium acetate, pH 6.0 (for endoglycosidase H) or 250 mM sodiumphosphate, pH 8.6 (for N-glycanase), and a final composition of 0.36%NaDodSO₄. 2.0% NP-40, 0.07 M β-mercaptoethanol. The samples were boiledagain for 1 min, endoglycosidase H or N-glycanase was added to a finalconcentration of 0.1 unit/ml or 20 units/ml, respectively, and thesamples were incubated 16 h at 37° C.

Treatment with endoglycosidase H resulted in slight but reproduciblechanges in the molecular weight of α-galactosidase A as judged by itsmigration in SDS gels, indicating the presence of mannoseoligosaccharide on α-galactosidse A. Treatment of the purified enzymewith N-glycanase produced a marked decrease in the apparent molecularweight of the enzyme, and converted the rather diffuse SDSpolyacrylamide band to a faster migrating and more compact band. Thesestudies indicate α-galactosidase A is glycosylated with complex mannosecontaining carbohydrate moieties.

EXAMPLE 5 Uptake of Recombinant α-galactosidase A by Normal and FabryFibroblasts

The uptake of the recombinant human α-galactosidase A by normal andFabry fibroblasts in culture was determined as follows.

Fibroblasts were obtained from Fabry patients (GM2775) or normalcontrols (GM0500B). Monolayers were allowed to grow to 80% confluency in25 cm² tissue culture flasks containing Delbecco's Modified Eagles (DME)Medium with 10% fetal bovine serum and antibiotics (penicillin at 100units/ml and streptomycin at 100 ξg/ml). Before recombinantα-galactosidase addition the monolayers were rinsed three times with DMEand recombinant α-galactosidase A was added to the medium. Flasks weregassed with 5% CO₂, sealed, and after incubation for 16 h at 27° C., themonolayers were rinsed twice with DME and trypsinized. Aftercentrifugation the cell pellet was resuspended in 0.5 ml of buffer Acontaining Triton X-100 (0.1%), and the cells were disrupted byfreeze-thawing three times. Protein and α-galactosidase A activity wereassayed in the resultant whole cell extract.

The results shown in Table 2 indicate that the recombinantα-galactosidase A is taken up in a dose dependent fashion that resultsin an approximately 59% increase in enzyme levels in normal fibroblasts,and a complete restoration of enzyme levels in Fabry fibroblasts.

TABLE 1 Enzyme Units Virus Transfer Vector Culture Media Cells AcCC1pCC4 2,301 830 AcCC2 pCC4 1,959 530 AcCC3 pCC5   29  2 AcMNPV None    2   0.2 Mock Infection None    3    0.4

TABLE 2 Enzyme Added Normal Cells Fabry Cells (unit/ml) (GM0500B)(GM2775) None 192  7 1000 221  59 2000 178 147 4000 306 270

What is claimed is:
 1. A method of treating a disease in a mammalresulting from deficiencies of α-galactosidase A comprisingadministering to said mammal a therapeutically effective amount of apharmaceutical composition comprising recombinant glycosylatedenzymatically active α-galactosidase A or an enzymatically activefragment thereof, prepared by a process comprising: (a) culturing a hostcell comprising a recombinant baculovirus comprising a nucleic acidencoding the precursor or mature form of an enzymatically activeα-galactosidase A; (b) recovering said recombinant glycosylatedenzymatically active human α-galactosidase A; and, optionally preparinga fragment thereof; and a pharmacologically acceptable carrier.
 2. Themethod of claim 1 wherein the disease is Fabry disease.
 3. The method ofclaim 1 wherein said effective amount comprises from about 50 to about10,000 units enzyme activity per kilogram body weight per day.
 4. Themethod of claim 1 wherein the active ingredients of the pharmaceuticalcomposition are administered in therapeutic amounts from about 0.1 μg toabout 2000 μg per kilogram body weight per day.
 5. The method of claim 4wherein the pharmaceutical composition is administered daily in severaldivided dosages.
 6. The method of claim 1 wherein the pharmaceuticalcomposition is administered orally, intravenously, intramuscularly,intranasally, intradermally, subcutaneously or as a suppository.
 7. Themethod of claim 1 wherein said pharmacologically acceptable carrier ispolyethylene glycol.